Cell shape, polarity and motility are controlled by the actin cytoskeleton. How F-actin filaments are assembled into functional arrays is still poorly understood. There are numerous F-actin binding proteins that are capable of cross-linking actin filaments and these proteins vary in the arrangement of filaments produced and the regulation of the binding activity. They can produce parallel, anti-parallel or orthogonal arrays of F-actin, but the complex interplay of these proteins is poorly understood. IN addition, a number of actin-binding proteins are regulated by factors such as Ca++ and the circumstance where this is important is not known. This proposal seeks to use molecular genetic and imaging approaches to continue to investigate the mechanisms used by cells to regulate the assembly of actin filament arrays. Myosin II has been found to be a major contributor to cytoskeletal integrity of Dictyostelium cells. The contribution of myosin II to forces applied to the surface will be investigated and compared to the contributions of other actin binding proteins using a flexible substratum assay. The contribution of myosin II to mammalian cell integrity will be investigated by using RNAi to inhibit the expression of myosin llB in tissue culture cells. Studies of ABP120 and alpha-actinin have shown that the actin binding domains of these proteins regulate their association with F-actin filaments. This has led to the hypothesis that not all actin filaments in cells are the same, since these proteins can distinguish between filaments in different parts of the cell. This important idea will be further investigated by studying the properties of the actin binding domains in vivo and in vitro. The role of Ca++ in regulating alpha-actinin localization will also be investigated in order to determine under what circumstances Ca++ regulation becomes important. Fimbrin is another member of this class of actin crosslinking proteins. There are two homologs of fimbrin in Dictyostelium cells, fimA and fimB. The fimA gene is similar to mammalian flmbrin having EF-hands and two actin-binding domains. FimB is a recently discovered homolog that lacks EF hands, but has PH domains and a talin homology domain. The function of each protein will be investigated by gene disruption, mutagenesis and localization techniques. The structure of the actin binding domains of each of these proteins will also be determined to investigate the mechanism by which they differentially recognize actin filaments.